Cyclooxygenase expression is elevated in retinoic acid-differentiated U937 cells

Cyclooxygenase (COX) is the rate-limiting enzyme for the biosynthesis of prostaglandins in monocytes/macrophages. The COX-1 is constitutively expressed in most tissues and may be involved in cellular homeostasis, whereas the COX-2 is an inducible enzyme that may play an important role in inflammation and mitogenesis. When U937 monocytic cells were incubated with retinoic acid (RA) for 48 h, cell differentiation took place with concomitant increases in prostaglandin E₂ (PGE₂) production and COX activity. In this study, the mechanism of RA (all-trans- or 9-cis-RA)-induced enhancement of PGE₂ biosynthesis in U937 cells was examined. Treatment of cells with all-trans- or 9-cis-RA up to 48 h caused an increase in PGE₂ production in a time- and dose-dependent manner. Both RA isomers caused the enhancement of PGE₂ production and the up-regulation of COX-1 expression at the protein and mRNA levels. The increase in COX-1 mRNA was found to precede the increase in COX-1 protein expression. Interestingly, the COX-2 protein and COX-2 mRNA were not detected in U937 cells, and their levels remained undetectable during the entire course of RA treatment. We conclude that treatment of U937 cells by RA for 48 h caused the initiation of cell differentiation, which was found to be concomitant with a significant increase in PGE₂ production mediated via the up-regulation of COX-1 mRNA and protein expression.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

Effects of AY 9944 on low density lipoprotein metabolism in cultured human fibroblasts

Treatment of cultured human fibroblasts with the hypocholesterolemic drug AY 9944 resulted in a marked increase in low density lipoprotein internalization and degradation for concentrations up to 5 X 10(-6)M. Low density lipoprotein binding was less affected. Concentrations above 5 X 10(-6)M resulted in a relative decrease in low density lipoprotein degradation, whereas binding and internalization plateaued. The stimulation of low density lipoprotein internalization took place within the first hours of incubation of cells with the drug, which suggests a direct effect on the cell membrane. Such phenomenon could account at least partially for the hypocholesterolemic effect of the drug, besides its inhibitory effect on 7-dehydrocholesterol reductase.     

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom

Colominic acid is an 2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A₂ activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.     

Reciprocal regulation of NADPH oxidases and the cyclooxygenase-2 pathway

The objective of this work was to analyze the possible association between cyclooxygenase-2 (COX-2) and NADPH oxidases (NOX) in liver cells, in response to various proinflammatory and toxic insults. First, we observed that treatment of Chang liver (CHL) cells with various COX-2 inducers increased reactive oxygen species (ROS) production concomitant with GSH depletion, phorbol 12-myristate 13-acetate (PMA) being the most effective treatment. Moreover, early changes in the oxidative status induced by PMA were inhibited by glutathione ethyl ester, which also impeded COX-2 induction. In fact, CHL cells expressed NOX1 and NOX4, although only NOX4 expression was up-regulated in the presence of PMA. Knock-down experiments suggested that PMA initiated a pathway in which NOX1 activation controlled COX-2 expression and activity, which, in turn, induced NOX4 expression by activation of the prostaglandin receptor EP4. In addition, CHL cells overexpressing COX-2 showed higher NOX4 expression and ROS content, which were decreased in the presence of the COX-2 inhibitor DFU. Interestingly, we found that addition of prostaglandin E(2) (PGE(2)) also induced NOX4 expression and ROS production, which might promote cell adhesion. Finally, we determined that NOX4 induction by PGE(2) was dependent on ERK1/2 signaling. Taken together, these results indicate that NOX proteins and COX-2 are reciprocally regulated in liver cells.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Antiprotozoal activity of bicyclic diamines with a N-methylpiperazinyl group at the bridgehead atom

ω-Aminoacyl and -alkyl derivatives of 4-(4-methylpiperazin-1-yl)bicyclo[2.2.2]octan-2-amines and of 5-(4-methylpiperazin-1-yl)-2-azabicyclo[3.2.2]nonanes were prepared and their activities were examined in vitro against the multiresistant K₁ strain of Plasmodium falciparum and against Trypanosoma brucei rhodesiense (STIB 900). Some of the newly synthesized compounds showed very promising antiprotozoal activity and selectivity. A few of the alkylamino-2-azabicyclo[3.2.2]nonanes exhibited high antiplasmodial activity, whereas a single bicyclo[2.2.2]octane derivative was the most potent antitrypanosomal compound. The results of the newly synthesized compounds were compared with the activities of already synthesized compounds and of drugs in use. Structure–activity relationships were discussed.     

Identification of galloylated propelargonidins and procyanidins in buckwheat grain and quantification of rutin and flavanols from homostylous hybrids originating from F. esculentumxF. homotropicum

The flavonoid rich grain of buckwheat (Fagopyrum esculentum Moench, Fam. Polygonaceae) is of high nutritional value. With the aim to improve its agronomic productivity, cultivars were crossed with the wild species F. homotropicum which, however, differs in its flavonoid content. The intention of this work was to determine the flavonoid composition in developed interspecific hybrids and to elucidate the proanthocyanidin structures. Seven compounds were purified from methanol extracts of buckwheat (Fagopyrum esculentum Moench) grains by Sephadex LH-20 column chromatography. Beside the procyanidin epicatechin-[4-8]-epicatechin-3-O-(3,4)-dimethylgallate the following propelargonidins were identified: epiafzelechin-[4-6]-epicatechin, epiafzelechin-[4-8]-epiafzelechin-[4-8]-epicatechin, epiafzelechin-[4-8]-epicatechin-3-O-(3,4-dimethyl)-gallate, epiafzelechin-[4-8]-epiafzelechin-[4-8]-epicatechin-3-O-(3,4-dimethyl)-gallate, epiafzelechin-[4-8]-epicatechin-3-O-4-methyl-gallate and epiafzelechin-[4-8]-epicatechin-p-OH-benzoate on the basis of HPLC and LC-MS/MS.     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

p-Hydroxyphenylpyruvate dioxygenase is a herbicidal target site for beta-triketones from Leptospermum scoparium

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism and is the molecular target site of β-triketone pharmacophores used to treat hypertyrosinemia in humans. In plants, HPPD is involved in the biosynthesis of prenyl quinones and tocopherols, and is the target site of β-triketone herbicides. The β-triketone-rich essential oil of manuka (Leptospermum scoparium), and its components leptospermone, grandiflorone and flavesone were tested for their activity in whole-plant bioassays and for their potency against HPPD. The achlorophyllous phenotype of developing plants exposed to manuka oil or its purified β-triketone components was similar to that of plants exposed to the synthetic HPPD inhibitor sulcotrione. The triketone-rich fraction and leptospermone were approximatively 10 times more active than that of the crude manuka oil, with I₅₀ values of 1.45, 0.96 and 11.5 μg mL⁻¹, respectively. The effect of these samples on carotenoid levels was similar. Unlike their synthetic counterpart, steady-state O₂ consumption experiments revealed that the natural triketones were competitive reversible inhibitors of HPPD. Dose-response curves against the enzyme activity of HPPD provided apparent I₅₀ values 15.0, 4.02, 3.14, 0.22 μg mL⁻¹ for manuka oil, triketone-rich fraction, leptospermone and grandiflorone, respectively. Flavesone was not active. Structure-activity relationships indicate that the size and lipophilicity of the side-chain affected the potency of the compounds. Computational analysis of the catalytic domain of HPPD indicates that a lipophilic domain proximate from the Fe²⁺ favors the binding of ligands with lipophilic moieties.     

Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.     

Synergism of Rana catesbeiana ribonuclease and IFN-gamma triggers distinct death machineries in different human cancer cells

Rana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-gamma. However, it is unclear how RC-RNase and RC-RNase/IFN-gamma induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-gamma-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-gamma induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-gamma can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity.     

The many faces of calmodulin in cell proliferation,programmed cell death,autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca^2 + receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.